The defective herpes simplex virus type 1 (HSV-1) DNA was physically mapped with restriction endonuclease (endo R) Bam HI and shown to consist of two unique fragments of identical molecular weight (1.2 megadaltons) and diffuse-microheterogenous fragments of 2 to 2.5 megadaltons. One of the small, unique fragments possessed the single endo R Eco RI site approximately 0.15 K base pairs from one end yielding a 1.1 megadalton fragment. Using "nick translated" defective DNA as a probe the origin of defective HSV-1 DNA was physically mapped in the right end 8 megadalton Hind III G fragment with endo R Bam HI.